Our studies are directed toward understanding the structure and function of human interferon-alphas and their receptors. The objectives of these studies is to delineate the rationale for the existence of this family of structurally-related proteins and to understand the mechanism by which they elicit their pleiotropic biological activities. Twenty-two components of human IFN-alpha derived from Sendai virus-induced human lymphoblastoid cells (Namalwa) were isolated by sequential monoclonal antibody affinity chromatography using four different monoclonal antibodies, ultrafiltration and reverse-phase HPLC. In addition, we have begun to employ the same techniques to isolate and characterize multiple components of human alpha interferon derived from human leukocytes isolated from peripheral blood in order to compare the biological and chemical properties of the differently derived interferon alpha components. Many biological properties of the human lymphoblastoid derived interferon have been examined. One component of interest, IFN-alpha o, exhibits high antiproliferative activity on Daudi and AU937 cells, but have low affinity for the IFN alpha 2b binding site on both cell lines. Partial amino acid sequence of IFN-alpha o revealed that it is indistinguishable from IFN-alpha 21. In order to study the relationship between component o and IFN-alpha 21, we cloned and expressed the IFN-alpha 21 gene in a pQE-30 expression system. The expressed protein was purified by metal chelate and 4F2 monoclonal antibody affinity chromatography. Biological functions of IFN-alpha 21 suggest that recombinant IFN-alpha 21 is indistinguishable from human lymphoblastoid IFN-alpha component o. In an effort to determine what domain of IFNA21 is responsible for its distinctive antiproliferative and binding activities, we have developed a DNA construct by incorporating a heterologous segment of IFNA21 into interferon-alpha 2.We will ascertain if the protein expressed from this recombinant DNA is biologically similar to IFNA21 and by using site directed mutagenesis, it will be possible to pinpoint the domain of interest. Results of biological activities of the purified IFN components mentioned above, as well as additional evidence from other laboratories, indicate that there are multiple binding sites and/or components to the Type I IFN cell surface receptor.We are now using the differences in competitive binding ability to investigate the multiple binding sites and isolate additional IFN binding components. Three monoclonal antibodies against a cloned Hu-IFN-alpha receptor have been used to purify several Type I IFN receptor associated components from human Daudi cells. We are presently studying these antibodies for their competitive binding and biological activities. We are also working to identify the components so far purified and study their ligand binding activities.Chemical characterization has always been a major program in our laboratory eg. N-terminal amino acid sequences. Currently, we are continuing our studies of the carbohydrate structure of the three major glycosylated human lymphoblastoid interferon alpha components in addition to determining the glycosylation of the human leukocyte interferon components.